gender: male strain: CD-1 age: 9 weeks tissue: liver treatment: exposed to BPA at 5000 µg/Kg/day
Treatment protocol
Five week-old mice were randomly divided into 3 groups (n = 6/group). Bisphenol-A was incorporated into a powedered diet (ingredients from SAFE Diets) containing (w/w) 49% starch, 14% casein, 2% cellulose, 24.4% sucrose, 2.5% arachis oil, 2.5% canola oil, 5% Minerals Mix (Ref 205b, SAFE), 0.5% Vitamins mix (Ref 200, SAFE) and 0.1% DL-methionine. Three diets were formulated to obtain an average BPA exposure of the animals of 0, 50 or 5000 µg/Kg/day. To calculate BPA exposure we considered a daily food consumption corresponding to 10% of body weight. Mice were exposed to the different diets for 28 days.
Growth protocol
Four week-old male CD-1 mice were purchased from Charles River (Les Oncins, France). Mice were acclimatized for one week, housed in polypropylene cages at 22+2°C on a 12 hour light/dark cycle and allowed free access to water (polypropylene bottles) and food. The in vivo study was conducted under the European Union Guidelines for the use and care of laboratory animals and were approved by an independent ethics committee.
Extracted molecule
total RNA
Extraction protocol
Immediately following euthanasia, liver was removed, weighted, dissected (~100 mg fragments from the large lobe), snap-frozen in liquid nitrogen and stored at -80°C until RNA extraction. Total RNA was extracted from frozen liver samples with TRIzol reagent (Invitrogen) following manufacturer's instructions. Total RNA samples were controlled for integrity on an Agilent 2100 Bioanalyzer (Agilent Technologies) and assayed at 260nm on a Nanodrop ND-1000 spectrophotometer (Thermo Scientific)
Label
Cy3
Label protocol
For each sample, Cyanine-3 (Cy3) labeled cRNA was prepared from 1 µg of total RNA using the One-Color Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Courtaboeuf, France). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
1.65 µg of Cy3-labelled cRNA (specific activity >13.8 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Oligo Microarrays (G4122F) enclosed in Agilent SureHyb-enabled hybridization chambers for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed sequentially in Wash buffer 1 (Agilent Technologies, 1 min), Wash buffer 2 (Agilent Technologies, 37°C, 1 min) and Stabilization and Drying Solution (Agilent Technologies, 30 sec).
Scan protocol
Slides were scanned immediately after washing on a GenePix™ 4000B array scanner
Data processing
The scanned images were analyzed with Feature Extraction Software 9.5 (Agilent) using default parameters (protocol GE1-v5_95 and Grid: 014868_D_20070207). All subsequent data analyses were done under R (www.r-project.org) using packages of Bioconductor (www.bioconductor.org). Raw data (median of pixels intensity) were imported into R using the read.maimages function from the limma package with the following weight function (assigning a weight of 1 or 0 to each spot): myfunw<-function(x) {okType<-x$ControlType==0 okFoundGreen<-x$gIsFound==1 okPos=x$gIsPosAndSignif==1 okWellAbove<- x$gIsWellAboveBG==1 as.numeric(okType & okFoundGreen & okPos & okWellAbove)} We selected the spots with a weight of 1 for 15 out of 18 microarrays or with a weight of 1 in all 6 microarrays from at least one experimental group. At this step, 23930 spots out of 45018 were selected. Data were then stored in an ExpressionSet object and normalized by the quantile method using the normalize.quantiles function from the preprocessCore library. Replicated probes on the array (identical ProbeName) were resolved by taking the median normalized signal of each set of replicated probes. The resulting matrix has 22514 rows each corresponding to a unique ProbeName (provided as data Matrix).
